Telomere is a structure formed of DNA repeat sequence (TTAGGG)n and some binding proteins at both ends of the eukaryotic chromosome. The telomere has the function of protecting the end of the chromosome from deterioration or from fusion, and it plays an important role in chromosomal location, replication, protection and in control of cell growth and survival, and is closely correlated with the cells apoptosis, transformation and immortalization. The telomere replication is accomplished by telomerase, a special reverse transcriptase, rather than by the typical DNA polymerase. In somatic cells of a normal person, as the expression of the telomerase is shut down, the telomere at the end of the chromosome is gradually shortened with cell division until the cell stops dividing or undergoes apoptosis. Therefore, a seriously shortened telomere is an indicator of cell aging. By virtue of the discovery about how chromosomes are protected by telomeres and telomerase, which reveals the mystery of human aging and suffering from serious diseases including cancer, Elizabeth Blackburn from the University of California (San Francisco, US), Carol Greider from Johns Hopkins University (US), and Jack Szostak from Harvard Medical School (US) and were awarded 2009 Nobel Prize in Physiology and Medicine.
The cancer cell has the property of indefinite division and proliferation, wherein the telomeric DNA is extended by reactivating the expression of the telomerase for most cases. However, the maintenance of the telomere length may also be realized through a telomerase-independent mechanism. In some cancer cells, the telomerase is still in an inhibitory state, wherein the telomeric DNA is extended through a “non-telomerase mechanism”, that is, Alternative Lengthening of Telomeres (ALT). The clinical research indicates that, in general, about 85-90% of the cancer cells are telomerase positive, and about 10-15% of the cancer cells are ALT positive, which means that this two mechanisms are alternative and the cancer cell is in a state of either this or that. For some cancers, the ALT positive rate is higher, for example, the ALT positive rate is nearly 60% for osteogenic sarcomas, nearly 40% for gastric cancers, about 30% for soft tissue sarcomas and astrocytoma, and about 25% for primary brain cancers and glioblastomas. More importantly, the cancer patient with positive ALT often has worse prognosis compared with that with positive telomerase. Therefore, the detection of ALT has a potential of being a reference for monitoring the disease condition and prognosis, and disease treatment. Theoretically, all the cancers can be detected by the combination of ALT detection and telomerase detection.
At present, the method for detecting and analyzing ALT mainly includes the following:
1. Determination of whether the telomere length of a cell can be maintained in the absence of the telomerase activity while the cells are successively divided and proliferated. This determination typically requires the successive division and subculture of the cells for 20-30 passages, and multiple detections of the telomerase activity and the telomere length, thus it is obviously time consuming and labor intensive.
2. Detection of the heterozygosity and fluctuation of the telomere length, this process can be accomplished by Southern blotting or FISH. The telomere length varies considerably among the ALT+ cell populations, wherein some cells have quite long telomere, and some have quite short telomere. The clinical specimen generally comprises a mixture of cancer cells/tissues and normal cells/tissues, and the telomere lengths in these two types of population are significantly different from each other, which causes difficulty to the determination of the ALT+ cells. Such detection suffers from complex operations and low sensitivity, for which at least 1000 cells are required.
3. Detection of ALT-related PML nucleosome. Copolymerization of telomeric DNA with PML protein is detected by probe hybridization in combination with immunofluorescence/immunohistochemistry. The PML nucleosome obtained from this copolymerization is one of the characteristics of ALT cells. Such detection suffers from complex operations and low sensitivity, for which about 1000 cells are required.
4. Detection of recombinant telomeric DNA structure with ALT characteristic. The T-loop structure formed by the recombination of ALT is detected by 2-D electrophoresis technology, which requires about 1×107 cells; and the C-loop is assayed by using a highly progressive φ29 DNA polymerase with the circular telomeric TC-ssDNA having characteristic of ALT as a template to synthesize high molecular weight telomeric TG-ssDNA, and the result is assayed by pulse electrophoresis and probe hybridization. Such detection has a sensitivity up to 1000 cells or higher, but requires the use of radioactive isotope, which is not suitable for clinical generalization.
TC-ssDNA (Telomeric C-single stranded DNA) is a single stranded DNA formed of the CCCTAA unit complementary to the repeat TTAGGG of telomeric G, which generally exists as an extrachromosomal circular DNA, is a specific marker of the ALT+ cells, and it does not exist in normal cells or telomerase positive cancer cells. Therefore, the presence of the ALT+ cells can be determined by detecting whether TC-ssDNA is present. PCR amplification has a quite high sensitivity in nucleic acid detection, so PCR amplification of TC-ssDNA will be a highly sensitive method for detecting ALT+ cells. However, because TC-ssDNA contains repeat CCCTAA sequence, and an upstream and a downstream primer recognition site are necessary for a PCR process, accordingly the TC-ssDNA cannot be used directly as a template for PCR amplification. In the present invention, the template probe is amplified by PCR by binding TC-ssDNA to the anchor probe and inhibiting the cleavage to the template probe through competitive binding, and the result of PCR amplification indicates the presence of TC-ssDNA, thereby the rapid and simple detection of the ALT+ cells with high sensitivity can be achieved.